International Journal of Systematic and Evolutionary Microbiology (2000), 50, 2075–2081
Printed in Great Britain
Evolutionary relationship between
dinoflagellates bearing obligate diatom
endosymbionts : insight into tertiary
endosymbiosis
Yuji Inagaki,1,2 Joel B. Dacks,2 W. Ford Doolittle,2 Kazuo I. Watanabe3
and Takeshi Ohama1
Author for correspondence : Yuji Inagaki. Tel : j1 902 494 2968. Fax : j1 902 494 1355.
e-mail : yinagai!is.dal.ca
1
JT Biohistory Research Hall,
1-1 Murasaki-cho,
Takatsuki, Osaka
569-1125, Japan
2
Program in Evolutionary
Biology, Canadian Institute
for Advanced Research,
Department of
Biochemistry and
Molecular Biology,
Dalhousie University,
Halifax, Nova Scotia,
Canada B3H 4H7
3
Department of Biology,
Faculty of Science, Osaka
University, Toyonaka,
Osaka 560-0043, Japan
The marine dinoflagellates Peridinium balticum and Peridinium foliaceum are
known for bearing diatom endosymbionts instead of peridinin-containing
plastids. While evidence clearly indicates that their endosymbionts are closely
related, the relationship between the host dinoflagellate cells is not settled. To
examine the relationship of the two dinoflagellates, the DNA sequences of
nuclear small-subunit rRNA genes (SSU rDNA) from Peridinium balticum,
Peridinium foliaceum and one other peridinin-containing species, Peridinium
bipes, were amplified, cloned and sequenced. While phylogenetic analyses
under simple models of nucleotide substitution weakly support the monophyly
of Peridinium balticum and Peridinium foliaceum, analyses under more
sophisticated models significantly increased the statistical support for this
relationship. Combining these results with the similarity between the two
endosymbionts, it is concluded that (i) the two hosts have the closest sister
relationship among dinoflagellates tested, (ii) the hypothesis that the diatom
endosymbiosis occurred prior to the separation of the host cells is most likely
to explain their evolutionary histories, and (iii) phylogenetic inferences under
complex nucleotide evolution models seem to be able to compensate
significant rate variation in the two SSU rDNA.
Keywords : Peridinium, plastid, SSU rDNA, among-site rate variation, nucleotide
substitution model
INTRODUCTION
It is well established that plastids (or chloroplasts) in
eukaryotes are derived either directly or indirectly
from highly reduced endosymbiotic cyanobacteria.
However, the evolution of extant photosynthetic
eukaryotes is quite complex (reviewed by Gray, 1992 ;
Gibbs, 1993 ; Delwiche & Palmer, 1997 ; Bhattacharya
& Medlin, 1998 ; Cavalier-Smith, 1999 ; Delwiche,
1999). Plastids surrounded by double membranes, so
.................................................................................................................................................
Abbreviations : Dist, distance ; indels, insertions/deletions ; ML, maximumlikelihood ; ML-Dist, maximum-likelihood distance ; MP, maximum-parsimony ; SSU rDNA, small-subunit rRNA gene ; kln L, log likelihood ; PINV,
proportion of invariable sites.
The GenBank accession numbers for the Peridinium balticum, Peridinium
foliaceum and Peridinium bipes SSU rDNA sequences are AF231803,
AF231804 and AF231805, respectively.
called ‘ primary ’ plastids, are found in green algae,
land plants, red algae and glaucocystophytes. These
primary plastids are believed to be evolved via direct
eukaryote–cyanobacterium endosymbiosis. It remains
unclear whether plastids in the four lineages are direct
descendants of a single endosymbiotic event. However,
recent evidence supports the monophyly of green algae
(plus land plants) and red algae, with weaker evidence
pointing also to a sister relationship with glaucocystophytes (Moreira et al., 2000).
‘ Secondary ’ plastids, be they triple or quadruple
membrane bound, are found in as widely divergent
eukaryotic lineages as euglenoids, apicomplexan parasites, chromist algae (heterokonts, haptophytes, cryptomonads and chlorarachniophytes) and dinoflagellates. This type of plastid is presumed to be derived
from a photosynthetic eukaryote bearing primary
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Y. Inagaki and others
plastids that was engulfed by a heterotrophic eukaryote through phagocytosis (or myzocytosis). Chlorarachniophytes and cryptomonads provide direct
evidence for this hypothesis (reviewed by Gilson &
McFadden, 1997) ; remnants of endosymbiotic nuclei
(nucleomorphs) and plastids are found in the periplastidal cytoplasm that is separated from the host
cytosol by two membranes. These cell structures have
been interpreted as intermediates in the reduction of
the engulfed photosynthetic eukaryotes into secondary
plastids without nucleomorphs. Recent molecular
phylogenetic studies have demonstrated that eukaryotes bearing secondary plastids usually have heterotrophic relatives, and they are most probably the
result of the independent acquisition of their plastids
after the separation of the photosynthetic lineage from
their heterotrophic sisters (Van de Peer et al., 1996 ;
Inagaki et al., 1997, 1998 ; Van der Auwera et al.,
1998).
In addition, several dinoflagellates maintain chromist
algae, which themselves have secondary plastids, as
endosymbionts (Delwiche, 1999). Such ‘ tertiary ’ endosymbioses are not as well studied, but the marine
dinoflagellates Peridinium balticum and Peridinium
foliaceum are well-established cases. The endosymbionts of Peridinium balticum and Peridinium foliaceum
are remarkably similar in their structural features and
pigment composition (reviewed by Chesnick et al.,
1997). The endosymbionts are separated from the host
cytosol by a single membrane, presumably having lost
their cell wall (Dodge, 1971 ; Tomas et al., 1973), and
the endosymbiont of Peridinium balticum undergoes
amitotic cell division (Tippit & Pickett-Heaps, 1976).
These observations strongly suggest that the algae are
not the transient association of the host cells with an
autonomous photosynthetic eukaryote. Given this, it
is surprising that the endosymbionts appeared to retain
their mitochondria as well as eukaryotic nucleus and
plastids (Dodge, 1971 ; Tomas et al., 1973).
Based on the photosynthetic pigmentation and recent
phylogenetic study of the nuclear small-subunit rRNA
(SSU rDNA) of the two endosymbionts, it was clearly
shown that the two endosymbionts are closely related,
and descendants of pennate diatoms (family Bacillariaceae) (Dodge, 1971 ; Tomas et al., 1973 ; Chesnick
et al., 1997). The phylogenetic analysis inferred from a
plastid gene, rbcL (encoding the large subunit of
ribulose-1,5-bisphosphate carboxylase\oxygenase),
also indicated that the endosymbiont of Peridinium
foliaceum has a diatom origin (Chesnick et al., 1996).
Curiously, despite the ultrastructural similarities of the
two hosts, Peridinium balticum and Peridinium foliaceum, their isozyme profiles are significantly different
(Whitten & Hayhome, 1986). These conflicting data
about their relationship make it difficult to determine
whether the obligate diatom endosymbiosis occurred
before or after the diversification of the host lineages.
To examine the phylogenetic positions of Peridinium
balticum and Peridinium foliaceum among dinoflagel2076
lates, we amplified, cloned and sequenced their (nuclear) SSU rDNA sequences, as well as the sequence of
an additional peridinoid species bearing a standard
(peridinin-containing) dinoflagellate plastid, Peridinium bipes. Our exhaustive phylogenetic analyses
inferred from SSU rDNA sequences succeeded in
recovering a closest sister relationship between Peridinium balticum and Peridinium foliaceum, and we
conclude that the ancestor of the two dinoflagellates
engulfed a pennate diatom.
METHODS
Amplification, cloning and sequencing of SSU rDNA. Peri-
dinium balticum (CS-38) and Peridinium foliaceum (LB1688)
were purchased from the CSIRO division of Marine Research, Australia (Castray Esplanade, Hobart, Tasmania
7000, Australia). Peridinium bipes (NIES 364) was purchased
from the National Institute for Environmental Studies,
Japan (16-2 Onogawa, Tsukuba, Ibaragi 305-0053, Japan).
Cells were cultured with the medium recommended by the
suppliers with 50 mg gentamicin mlV" to prevent bacterial
growth. One gram of frozen cells was ground into a fine
powder using a bead-mill and resuspended in one volume of
CTAB extraction buffer [2 % (w\v) cetyltrimethylammonium bromide (CTAB), 100 mM Tris\HCl (pH 8n0), 20 mM
EDTA (pH 8n0), 1n4 M NaCl]. Nucleic acids were extracted
twice with chloroform following incubation for 1 h at 65 mC,
and then precipitated by addition of an equal volume of
CTAB precipitation buffer [1 % (w\v) CTAB, 50 mM
Tris\HCl (pH 8n0), 10 mM EDTA (pH 8n0)]. The resultant
pellet was resuspended in high-salt buffer [10 mM Tris\HCl
(pH 8n0), 0n1 mM EDTA (pH 8n0), 1 M NaCl]. After 2propanol precipitation, nucleic acids were dissolved in
distilled water and used for PCR.
A pair of synthesized primers, 5h-TACCTGGTTGATCC
TGCCAGTA-3h and 5h-CCATCCGCAGGTTCACCTCA3h, was used to amplify nearly the entire SSU rDNA. PCR
was carried out as follows : denaturation at 94 mC for 15 s,
annealing at 60 mC for 1 min and extension at 72 mC for
2 min for 30 cycles. The amplified fragments were subsequently ligated into the pT7Blue T-vector (Novagen) and
transformed into Escherichia coli JM109. DNA sequences
were determined for both strands by cycle sequencing with
dye-terminators using a DNA sequencer model 377 (Applied
Biosystems).
Phylogenetic analyses. The three Peridinium rDNA sequences were manually aligned along with the previously
published sequences of 49 dinoflagellates and two apicomplexan parasites (Perkinsus sp., L07375 ; Toxoplasma gondii,
X65508) using  version 4.0b12 (Maddison &
Maddison, 2000). For alignment editing, we referred to the
secondary structure of SSU rRNA molecule deposited in the
rRNA WWW Server at the University of Antwerp (http :\\
rrna.uia.ac.be\index.html). After polishing of the alignment
by eye and exclusion of all ambiguous sites, it included 54
taxa with 1723 sites. Preliminary phylogenetic analyses were
performed on this dataset under maximum-parsimony (MP)
optimality criteria. Subsequently we generated an additional
two datasets for the later analyses.
A second alignment comprising 40 taxa and 1723 sites was
generated by removing redundant taxa (i.e. taxa in the clades
with bootstrap scores of 100 in MP analyses). This dataset
was tested for optimal fit of various models of nucleotide
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Relationship between P. balticum and P. foliaceum
evolution using  version 3.0b3 (Posada &
Crandall, 1998). The proportion of invariable sites (PINV), a
discrete γ distribution (four categories) and base frequencies
were similarly estimated from the dataset. A maximumlikelihood distance (ML-Dist) bootstrap analysis (100 resampling) under an optimal model of nucleotide substitution
incorporating the PINV and a discrete γ distribution with the
empirical base frequencies was performed. For each replicate, starting trees were constructed by 100 random
additions.
the four Peridinium SSU rDNA, we measured the
number of unique substitutions among 1730 sites in
each sequence (Table 1). Interestingly, the Peridinium
foliaceum gene appeared to have 73 unique substitutions, almost twice that observed in the other three
taxa (Table 1). These observations strongly imply that
the Peridinium foliaceum SSU rDNA evolves almost
twice as fast as the Peridinium balticum gene.
The third dataset of 22 taxa and 1718 sites was subjected to
analysis using  (Posada & Crandall, 1998), and
analysed using maximum-likelihood (ML) and distance
(Dist) methods under two optimal models recommended as
well as three simple models. Bootstrap analyses (100 resampling) using the Dist method under the five models were performed. ML bootstrap analyses under the two optimal
models (100 resamplings) were operated without branchswapping (the ‘ Fast ’ stepwise-addition option in *
version 4.0b3 ; Swofford, 1998) to save computational time.
For all phylogenetic analyses in this study, * version
4.0b3 was used (Swofford, 1998).
Phylogenetic analyses
RESULTS
The Peridinium SSU rDNA sequences
The amplified regions of SSU rDNA for Peridinium
bipes, Peridinium balticum and Peridinium foliaceum
were 1796, 1796 and 1795 bp, respectively. GjC
contents are 48, 47 and 46 mol %, respectively. The
partial sequence of the previously reported SSU rDNA
for Peridinium foliaceum (M88517) was identical to the
sequence determined in this study. The number of
nucleotide substitutions and single nucleotide insertions\deletions (indels) between the Peridinium balticum and Peridinium foliaceum genes were 145 and 4,
respectively (Table 1), while the endosymbiont SSU
rDNAs have only 1 % difference (18 substitutions out
of 1788 sites ; Chesnick et al., 1997). No difference
between the putative secondary structures of the
Peridinium balticum and Peridinium foliaceum rDNAs
was detected (data not shown). Based on their structures, 103 out of 145 substitutions and two of the indels
corresponded to non-conserved regions. Comparing
Table 1. Nucleotide substitution matrix of the four
Peridinium SSU rDNA and number of unique
substitutions
.................................................................................................................................................
Pfol, Peridium foliaceum ; Pbal, Peridinium balticum ; Pbip,
Peridinium bipes ; Psp., Peridinium sp. Number of single
nucleotide insertions\deletions are indicated in parentheses.
Pfol
Pbal
Pbip
Psp.
Pbal
Pbip
Psp.
Unique substitutions*
145 (4)
223 (0)
175 (2)
233 (1)
141 (5)
108 (1)
73
34
28
36
* Includes unique insertions\deletions.
In preliminary MP analyses, Peridinium balticum and
Peridinium foliaceum were positioned as sister taxa
(bootstrap score, 35 ; data not shown). Corresponding
to the outstanding number of the unique substitutions
found in the Peridinium foliaceum gene (Table 1), the
Peridinium foliaceum branch was twice as long as that
of Peridinium balticum in the most parsimonious trees
(data not shown, but the same results were observed in
Fig. 1). Recently it has been shown that phylogenetic
inference without accounting for among-site rate
variation can lead to artefactual results (e.g. Silberman
et al., 1999 ; Stiller & Hall, 1999). Because the difference
in substitution rate between the SSU rDNA sequences
of interest may mask phylogenetic signal, it is possible
that our MP analyses may not be able to robustly
reconstruct relationships.
A subdataset (40 taxa\1723 sites), which does not
include redundant taxa, was analysed using 
(Posada & Crandall, 1998). Through calculation of
log-likelihood (kln L) scores, this program found that
a TrN model of nucleotide evolution (Tamura & Nei,
1993) incorporating the proportion of invariable sites
and a discrete γ distribution (four categories) (TrNj
PINVjγ) was significantly better than other models
examined. The dataset was therefore analysed using a
ML-Dist method under this model (Fig. 1a). For the
most part, the resultant tree corresponds to the general
classification and previous investigations of dinoflagellate phylogeny based on SSU rDNA sequences (e.g.
Grzebyk et al., 1998 ; Montresor et al., 1999). Our
analysis showed weak affinity for the Peridinium
balticum–Peridinium foliaceum clade and strong support for the Peridinium bipes–Peridinum sp. relationship (bootstrap scores, 54 and 100, respectively ; Fig.
1a).
Considering computational time, for this set of analyses, a smaller dataset (20 taxa\1718 sites) was generated.  (Posada & Crandall, 1998) found that
TrN (Tamura & Nei, 1993) and general-time-reversible
(GTR) models (Rodrı! guez et al., 1990) incorporating
the PINV and a discrete γ distribution best describe the
dataset (kln L scores estimated under the two models
are almost the same, 10782). The kln L scores under
the simple models, JC (Jukes & Cantor, 1969), F81
(Felsenstein, 1981) and K2P (Kimura, 1980), that
incorporated neither PINV and a discrete γ distribution,
were poor (kln L l 12146, 12126 and 11811, respectively). Phylogenies inferred from the third dataset
matched the overall topology of those in the first two
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Y. Inagaki and others
.....................................................................................................
Fig. 1. Phylogenetic trees inferred from
dinoflagellate SSU rDNA sequences. (a) The
maximum-likelihood distance (ML-Dist) tree
(40 taxa/1723 sites) reconstructed under a
TrN nucleotide substitution model (Tamura
& Nei, 1993) incorporating the proportion of
invariable sites (PINV) and a discrete γ
distribution (four categories) (TrNjPINVjγ).
For the bootstrap analyses, 100 replicates of
the original dataset were used, with starting
trees for each replicate constructed by 100
random addition. Only bootstrap scores
above 50 % are indicated. (b) The best
maximum-likelihood (ML) tree inferred from
the dataset (20 taxa/1718 sites) under a
TrNjPINVjγ model. The bootstrap scores
indicated in roman and italic were calculated by ML-Dist and ML methods under
a TrNjPINVjγ model (100 resamplings),
respectively. Only bootstrap score above
50 % are indicated. For the Peridinium
balticum–Peridinium foliaceum clade, bootstrap scores obtained from ML-Dist and ML
analyses under a general-time-reversible
(GTR) model (Rodrı! guez et al., 1990)
incorporating the PINV and a discrete γ
distribution (four categories) are also
indicated. ML bootstrap analyses were
conducted with no branch-swapping using
the ‘ Fast ’ stepwise-addition option in PAUP*
version 4.0b3 (Swofford, 1998). Lineages
bearing tertiary plastid are highlighted by
arrows. The Gymnodinium breve, Gymnodinium galatheanum and Gymnodinium
aureolum SSU rDNA are not included in these
analyses.
(Fig. 1b). For the Peridinium balticum–P. foliaceum
clade, bootstrap scores of 62 and 78 were obtained
using ML-Dist methods under TrNjPINVjγ and
GTRjPINVjγ models, respectively. The ML bootstrap analyses under those two optimal models gave
bootstrap scores of 77 and 74 for the same clade (Fig.
1b ; Table 2). All of the top 100 ML trees reconstructed
under the optimal models included the Peridinium
2078
balticum–Peridinium foliaceum clade (data not shown).
However, neither Dist or ML analyses under the
simple models, JC, F81 and K2P, supported this clade
(Table 2). While the Peridinium bipes–Peridinium sp.
clade was solid irrespective of the methods or models
(Table 2), we failed to detect any connection between
the Peridinium balticum–Peridinium foliaceum and
Peridinium bipes–Peridinium sp. clades (Fig. 1).
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Relationship between P. balticum and P. foliaceum
Table 2. Impact of nucleotide substitution and among-site rate variation models on dinoflagellate phylogeny
.................................................................................................................................................................................................................................................................................................................
Symbols\abbreviations : j, the best ML tree includes the topology of interest ; k, the bootstrap consensus tree or best ML tree
does not have the topology of interest ; , bootstrap analysis using ML method under JC, F81 or K2P model was not done. ML
bootstrap analyses were conducted with no branch-swapping (* version 4.0b3 ; Swofford, 1998).
Criteria
Peridinium balticum–Peridinium foliaceum clade
Peridinium bipes–Peridinium sp. clade
Basal branching of Gonyaucales and Nocticales
Model
Dist (bootstrap)
ML (bootstrap)
best ML tree
Dist (bootstrap)
ML (bootstrap)
best ML tree
Dist (bootstrap)
ML (bootstrap)
best ML tree
DISCUSSION
Impact of nucleotide evolution models on
dinoflagellate SSU rDNA phylogeny
Our SSU rDNA analyses reconstruct major dinoflagellate clades, i.e. the clades comprised of the species
belonging to the families Gonyaulacales, the Symbiodinium and Gymnodinium species, the two Gymnodinium species and Lepidodinium viride (Fig. 1), in
agreement with other previous studies (e.g. Grzebyk et
al., 1998 ; Montresor et al., 1999). In phylogenetic
inferences under simple nucleotide substitution models
without accounting for among-site rate variation,
species belonging to the families Gonyaulacales and
Nocticales, such as Alexandrium minutum, Alexandrium margalefeii, Ceratium tenue, Crypthecodinium
cohnii, Amphidinium belauense and Noctiluca scintillans, were found as basal branches among dinoflagellates (Table 2). However, in our phylogenies reconstructed under the complex models, which best described our SSU rDNA dataset, there is no statistical
support for relative branching order between the
robust clades that we obtained, nor for the deeply
diverging branches in our trees (Fig. 1 ; Table 2).
Considering their long branch lengths, we suspect the
positions of some of the basal diverging dinoflagellate
sequences, in particular the family Gonyaulacales,
may be artefactual, resulting from ‘ long branch
attraction ’. Our analyses suggest that the complex
models seem less susceptible to artificial resolution due
to long branch attraction as shown by Silberman et al.
(1999).
‘ Single-endosymbiosis ’ versus ‘ separateendosymbiosis ’
Despite both morphological and molecular evidence
strongly indicating a close relationship between the
diatom endosymbionts of Peridinium balticum and
Peridinium foliaceum, the available data on the dino-
JC
F81
K2P
TrNjPINVjγ
GTRjPINVjγ
k

k
100

j
79

j
k

k
100

j
74

j
k

k
100

j
74

j
62
77
j
100
100
j
k
k
k
78
74
j
100
100
j
k
k
k
flagellate hosts themselves are not sufficient to resolve
their relationships (reviewed by Chesnick et al., 1997).
This relationship is the key to the history of plastid
acquisition in these two Peridinium species. So far,
isozyme profile (Whitten & Hayhome, 1986) is consistent with the ‘ separate-endosymbiosis ’ hypothesis –
that the two dinoflagellates captured the same or a
similar diatom species independently. However,
no molecular sequence data have been available for
both Peridinium balticum and Peridinium foliaceum
host cells, and statistical examinations of their relationship were therefore impossible. If a phylogenetic
affinity between the two hosts was statistically supported, the ‘ single-endosymbiosis ’ hypothesis – that
the ancestor of two dinoflagellates engulfed a diatom
would be favoured over the ‘ separate-endosymbiosis ’
hypothesis.
Preliminary MP analyses lent no support to either
acquisition scenarios of the diatom endosymbionts in
Peridinium balticum and Peridinium foliaceum. However, as these analyses did not account for rate
heterogeneity among sites, more stringent analyses
with biologically relevant models for nucleotide evolution were performed. Analyses using ML-Dist and
ML methods under the complex models of nucleotide
evolution significantly improved the statistical support
for the Peridinium balticum–Peridinium foliaceum clade
(Table 2). These results are in contrast to inferences
under simple models without consideration of amongsite rate variation (Table 2). Difficulty in resolving the
relationship between Peridinium balticum and Peridinium foliaceum is most probably caused by the
variation of nucleotide substitution rates between the
two SSU rDNA, and thus biologically relevant nucleotide evolution models should be adopted to deal with
this problematic dataset. Since these models were
found to best describe the dataset, we conclude that
Peridinium balticum and Peridinium foliaceum are the
closest relatives among the species examined. This is in
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Y. Inagaki and others
agreement with morphological comparisons of relevant cell structure (reviewed by Chesnick et al., 1997).
Large indels in SSU rDNA are presumed to accelerate
the substitution rate over the entire gene in order to
preserve overall rRNA structure (Stiller & Hall, 1999).
Unfortunately, comparison between the putative secondary structures of the Peridinium balticum and
Peridinium foliaceum SSU rDNA suggests no such
indels (data not shown). We can see no obvious
molecular or biological rationale for the rate acceleration of the Peridinium foliaceum SSU rDNA.
Further sequencing and comparison of the Peridinium
balticum and Peridinium foliaceum protein-coding
genes are necessary to examine whether the rate
acceleration extends to the entire nuclear genome of
Peridinium foliaceum. If so, this might reconcile their
isozyme profiles (Whitten & Hayhome, 1986) that
seem contradictory to the conclusions reached by
morphological studies and our molecular analyses.
Assembled, these data lead us to prefer the scenario
that a common ancestor of the two dinoflagellates
engulfed a pennate diatom.
Nevertheless, the relationship between Peridinium
balticum and Peridinium foliaceum still needs to be
tested by SSU rDNA phylogeny with a broader taxon
sampling. If some species with peridinin-containing
plastids directly clusters with either Peridinium balticum or Peridinium foliaceum in a future study, the
‘ single-endosymbiosis ’ scenario will be disfavoured.
Frequency of tertiary endosymbiosis in dinoflagellate
evolution
When and how many times tertiary endosymbioses
have occurred during dinoflagellate evolution is an
intriguing question. The relative positions of the
tertiary plastid-containing dinoflagellates might give
some indication of the number of times these events
have occurred. Unfortunately, the relative position of
the Peridinium balticum–Peridinium foliaceum clade in
the dinoflagellate phylogeny is totally unresolved ; this
clade showed no affinity to other species including
Peridinium sp. and Peridinium bipes (Fig. 1). Kishino–
Hasegawa tests rejected neither the monophyly of the
four Peridinium species nor the basal separation of the
Peridinium balticum–Peridinium foliaceum clade (data
not shown). However, the triple-enveloped eyespots in
the cytosols of Peridinium balticum and Peridinium
foliaceum have been described, and presumed to be
vestiges of plastids surrounded by three membranes
(Horiguchi & Pienaar, 1991). Therefore, the ancestor
of the two dinoflagellates may have replaced the
original plastid with a diatom endosymbiont, arguing
against the diatom endosymbiosis as the basal condition for the dinoflagellate clade.
Recently, the three marine dinoflagellates Dinothrix
paradoxa, Gymnodinium quadrilobatum and Peridinium quinquecorne have been described as bearing
diatom endosymbionts (Horiguchi & Pienaar, 1991,
1994 ; Horiguchi & Chihara, 1993). Unfortunately,
2080
whether these diatoms are permanent residents is
unclear. Cavalier-Smith & Lee (1985) make the distinction between transient endosymbiont and permanent resident (organelle) based on extent of hostendosymbiont integration. Organelles transfer some of
their genes to their host’s nucleus, and possess the
molecular machinery to translocate the proteins encoded by such genes. Searching the nuclear genomes of
diatom-bearing dinoflagellates for genes that are unambiguously derived from the diatom endosymbiont
might clarify the question of whether the endosymbionts deserve true organelle status. The relationships among the dinoflagellates bearing diatom endosymbionts, including Peridinium balticum and Peridinium foliaceum, and their relative positions in dinoflagellate phylogeny are important in estimating the
frequency of tertiary endosymbiosis between diatoms
and dinoflagellates.
The extant plastid in the dinoflagellate Lapidodinium
viride is most probably acquired by plastid replacement
via tertiary endosymbiosis (reviewed by Delwiche,
1999). This dinoflagellate possesses a green-pigmented
plastid surrounded by four membranes. In our analyses, Lapidodinium robustly clustered with Gymnodinium catenatum and Gymnodinium fuscum, which
have peridinin-containing plastids (Fig. 1). Therefore,
plastid replacement occurred after the separation of
Lapidodinium and the two Gymnodinium species. Other
tertiary plastids, assumed to be originated from haptophytes, have been reported in Gymnodinium breve,
Gymnodinium galatheanum and Gymnodinium aureolum (Delwiche, 1999). Then we presume that tertiary
endosymbioses (and plastid-replacement) have occurred at least three times during dinoflagellate evolution
(highlighted by arrows in Fig. 1 ; Gymnodinium breve,
Gymnodinium galatheanum and Gymnodinium aureolum are not included).
Dinoflagellates are easily maintained in the laboratory,
are experimentally tractable, and have had a complex
and varied history of plastid acquisition. They therefore make excellent models for the investigation of
tertiary endosymbiosis. In order to best study this
fascinating problem, however, a robust phylogeny of
dinoflagellates based both on biologically relevant
models of evolution for SSU rDNA as well as analyses
from protein-coding genes are crucial.
ACKNOWLEDGEMENTS
We thank M. Ehara (Kobe University, Japan) for valuable
discussions and comments on the previous version of this
manuscript. We also thank J. M. Archibald, A. Lohan and
A. J. Roger (Dalhousie University, Canada) for critical
comments on this manuscript. Y. I. and K. I. W. are supported by a Research Fellowship of the Japanese Society for
Promotion of Science for Young Scientists in Abroad.
J. B. D. is supported by a Walter C. Sumner Memorial
Fellowship and a studentship awarded to W. F. D. by the
Canadian Medical Research Council. W. F. D. is supported
by grant ML4465 from the Canadian Medical Research
Council. A visiting associate professor position was arranged
International Journal of Systematic and Evolutionary Microbiology 50
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Relationship between P. balticum and P. foliaceum
for T. O. at Osaka University in order to permit collaboration with postgraduates and use of the various facilities.
phytes and dinoflagellates as inferred from mitochondrial
sequences. J Mol Evol 45, 295–300.
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